Recombinant Lactobacillus plantarum Phosphoribosyl-ATP pyrophosphatase (hisE)

How can I express recombinant hisE from Lactobacillus plantarum in a heterologous system?

For heterologous expression of Lactobacillus plantarum genes, E. coli is often the preferred expression system due to its rapid growth and well-established protocols. A methodological approach involves:

• PCR amplification of the hisE gene from L. plantarum genomic DNA using specific primers with appropriate restriction sites

• Cloning the amplified gene into an expression vector (pET series vectors are commonly used)

• Transformation into an E. coli expression strain (BL21(DE3) or similar)

• Induction of protein expression using IPTG at optimized conditions (typically 0.5-1 mM IPTG at 16-37°C)

• Cell harvesting and protein purification using affinity chromatography (if a His-tag or other tag was incorporated)

Similar approaches have been successfully employed for other L. plantarum enzymes, such as the heterologous expression of the iunH gene in E. coli, which resulted in complete degradation of nucleosides after 3 hours .

What are the optimal conditions for preserving hisE enzyme activity during purification?

To maintain optimal activity of recombinant Phosphoribosyl-ATP pyrophosphatase during purification:

• Use a buffer system with pH 7.0-7.5, as many Lactobacillus enzymes show optimal activity in this range

• Include protective agents such as 1-5 mM DTT or β-mercaptoethanol to prevent oxidation of cysteine residues

• Add 10-20% glycerol to stabilize the enzyme during storage

• Maintain low temperatures (4°C) throughout the purification process

• Consider adding protease inhibitors to prevent degradation

• For long-term storage, flash-freeze aliquots in liquid nitrogen and store at -80°C

These conditions are similar to those used for preserving activity of other nucleoside-metabolizing enzymes from Lactobacillus, which have been shown to retain their function under controlled laboratory conditions .

How can I verify the functional activity of recombinant hisE from L. plantarum?

To verify the functional activity of recombinant hisE:

• Enzymatic assay: Measure the hydrolysis of phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate using HPLC or coupled enzyme assays

• Complementation studies: Use the recombinant enzyme to complement a hisE-deficient bacterial strain and assess restoration of histidine prototrophy

• Comparative analysis: Compare the kinetic parameters (Km, Vmax) with those of hisE enzymes from other organisms

• Structural integrity assessment: Use circular dichroism spectroscopy to confirm proper protein folding

• Thermal shift assays: Evaluate protein stability under various conditions

Similar verification approaches have been used for other L. plantarum enzymes, such as the nucleoside hydrolase iunH, where functional confirmation was achieved through both heterologous expression and gene knockout experiments .

What gene knockout strategies are most effective for studying hisE function in Lactobacillus plantarum?

Effective gene knockout strategies for L. plantarum include:

• CRISPR-Cas9 system: Design guide RNAs targeting the hisE gene region and transform with a CRISPR-Cas9 plasmid

• Homologous recombination: Create a construct with upstream and downstream homology regions flanking an antibiotic resistance marker

• Temperature-sensitive plasmid integration: Use plasmids that can integrate at non-permissive temperatures and select for double crossover events

• Single-crossover disruption: Insert a non-replicating plasmid into the target gene via homologous recombination

Verification of knockout should include PCR confirmation, sequencing, and phenotypic analysis. In studies with L. plantarum SQ001, gene knockout experiments were successfully used to demonstrate a 50% reduction in nucleoside degradation upon iunH gene knockout, confirming its role in nucleoside metabolism .

How does hisE expression in L. plantarum change under different growth conditions?

Expression of metabolic enzymes like hisE in L. plantarum typically varies with growth conditions:

• Nutrient availability: Expression increases in histidine-limited environments and decreases when histidine is abundant

• Growth phase: Expression patterns may differ between exponential and stationary phases

• pH changes: Acidic environments often alter expression of biosynthetic enzymes

• Temperature stress: Heat or cold shock can induce changes in expression

• Carbon source: Different carbon sources can trigger metabolic shifts affecting amino acid biosynthesis

How can I engineer L. plantarum strains with enhanced hisE activity for biotechnological applications?

Engineering L. plantarum for enhanced hisE activity involves:

• Promoter engineering: Replace the native promoter with stronger constitutive or inducible promoters

• Codon optimization: Adjust the coding sequence to use preferred codons in L. plantarum

• Directed evolution: Create libraries of hisE variants and screen for improved activity

• Protein engineering: Introduce specific mutations based on structural analysis to enhance catalytic efficiency

• Metabolic flux optimization: Modify related pathways to increase substrate availability

• Multi-copy integration: Introduce multiple copies of the optimized gene

These approaches require sophisticated genetic tools but can yield strains with significantly enhanced enzymatic activities. Similar engineering approaches have been used with other L. plantarum enzymes involved in nucleoside metabolism .

What is the relationship between hisE activity and nucleoside metabolism in Lactobacillus plantarum?

While hisE is primarily involved in histidine biosynthesis, there are potential interactions with nucleoside metabolism pathways:

Genome analysis of L. plantarum has revealed interconnected metabolic networks. In L. plantarum SQ001, genes involved in nucleotide transport and metabolism constitute a significant portion of the genome, with multiple genes identified for nucleoside hydrolases (iunH), ribonucleoside hydrolases (rihA, rihC), and nucleoside permease (yxjA) .

How do post-translational modifications affect hisE function in L. plantarum compared to recombinant versions?

Post-translational modifications (PTMs) can significantly impact enzyme function:

• Phosphorylation: May regulate enzyme activity through conformational changes

• Acetylation: Can affect protein stability and interaction with other proteins

• Oxidation: Cysteine residues may form disulfide bonds affecting structure

• Proteolytic processing: N-terminal or C-terminal cleavage might occur

• Glycosylation: Though rare in bacteria, some surface proteins can be glycosylated

To study these differences:

• Compare native and recombinant enzyme kinetics

• Use mass spectrometry to identify PTMs

• Create site-directed mutants at potential modification sites

• Perform activity assays under different redox conditions

Studies on L. plantarum enzymes have shown that environmental conditions can affect protein function through various mechanisms, which should be considered when working with recombinant versions .

What are the common pitfalls in purifying recombinant L. plantarum hisE and how can they be overcome?

Common purification challenges include:

• Low expression levels: Optimize codon usage or try different expression vectors and host strains

• Inclusion body formation: Lower induction temperature (16-20°C), reduce IPTG concentration, or use solubility-enhancing tags

• Protein instability: Add stabilizing agents like glycerol or specific ions that may enhance stability

• Co-purifying contaminants: Use additional purification steps such as ion exchange or size exclusion chromatography

• Loss of activity during purification: Minimize exposure to room temperature and optimize buffer conditions

Successful purification strategies should be validated by SDS-PAGE analysis, western blotting, and activity assays. Similar strategies have been employed for purifying nucleoside hydrolases from L. plantarum, which demonstrated complete nucleoside degradation in biochemical assays .

How can I design specific inhibitors for L. plantarum hisE to study its role in metabolic pathways?

Designing specific inhibitors involves:

• Structure-based design: Use computational modeling based on crystal structures or homology models

• High-throughput screening: Test libraries of compounds for inhibitory activity

• Substrate analogs: Synthesize molecules that mimic the natural substrate

• Fragment-based approach: Identify small molecules that bind to the active site and then link or grow them

• Natural product screening: Test compounds from natural sources for inhibition

Testing approaches include:

• In vitro enzyme assays with purified protein

• Cell-based assays measuring growth in histidine-limited media

• Metabolomic analysis to detect pathway intermediates

• Thermal shift assays to confirm binding

Understanding enzyme mechanisms, such as those elucidated for the nucleoside hydrolase in L. plantarum SQ001, can provide valuable insights for inhibitor design strategies .

What bioinformatic approaches are most useful for analyzing hisE sequence conservation and predicting functional domains?

Effective bioinformatic approaches include:

• Multiple sequence alignment: Align hisE sequences from diverse bacterial species to identify conserved residues

• Phylogenetic analysis: Construct trees to understand evolutionary relationships

• Protein domain prediction: Use tools like Pfam, SMART, or InterPro to identify functional domains

• Structural modeling: Generate homology models using tools like SWISS-MODEL or AlphaFold

• Molecular dynamics simulations: Predict protein behavior and substrate interactions

• Coevolution analysis: Identify residues that evolve together, suggesting functional relationships

These approaches can reveal important information about catalytic residues, substrate binding sites, and regulatory regions. Genome analysis of L. plantarum SQ001 employed similar bioinformatic approaches to characterize its 3,549,454 bp genome containing 3361 coding sequences and identify key metabolic genes .

How might CRISPR-Cas gene editing be optimized for studying hisE function in L. plantarum?

Optimizing CRISPR-Cas for L. plantarum involves:

• Vector design: Develop plasmids with appropriate replication origins and selection markers for L. plantarum

• Guide RNA selection: Design sgRNAs with high specificity and low off-target effects

• Delivery methods: Optimize electroporation protocols specifically for L. plantarum

• Cas9 variants: Test different Cas9 proteins (SpCas9, SaCas9) or nuclease-deficient variants for CRISPRi

• Repair template design: Create efficient templates for homology-directed repair

• Screening strategies: Develop high-throughput methods to identify successful edits

Recent advances in bacterial CRISPR technologies can be adapted for L. plantarum, enabling precise genetic manipulation. Similar genetic engineering approaches have been used to study gene function in L. plantarum, as demonstrated with the iunH gene knockout experiments .

What role might hisE play in L. plantarum's ability to colonize different host environments?

The role of hisE in colonization may involve:

• Nutritional adaptation: Enabling growth in histidine-limited environments

• Stress response: Contributing to survival under acidic conditions or nutrient limitation

• Host interaction: Affecting bacterial surface properties that influence adhesion

• Biofilm formation: Participating in metabolic networks supporting community development

• Competitive fitness: Providing growth advantages in competition with other microbes

Research approaches to investigate this include:

• Comparing colonization efficiency of wild-type and hisE-deficient strains

• Analyzing hisE expression in different host environments

• Studying metabolic profiles during colonization

Studies with L. plantarum SQ001 demonstrated that it can successfully colonize the gut and improve microbial community composition, highlighting the importance of understanding metabolic adaptations in host colonization .

How can systems biology approaches integrate hisE function within the broader metabolic network of L. plantarum?

Systems biology approaches include:

These approaches can reveal emergent properties and non-obvious interactions. The analysis of L. plantarum SQ001 demonstrated interconnections between nucleoside metabolism and other pathways, revealing that these bacteria not only metabolize nucleosides but also regulate host UA metabolism through multiple mechanisms, including modulation of xanthine oxidase and renal transport proteins GLUT9 and ABCG2 .